Tissue-specific gene expression is largely regulated at the level of transcription through the combinatorial action of multiple tissue-restricted activators. Many liver-specific genes, whose products play critical roles in metabolism and homeostasis, are under the control of FoxA proteins. These include apolipoprotein A1, the major protein component of HDL, whose plasma concentrations correlate inversely with incidence of atherosclerosis, one of the disorders of the metabolic syndrome. This proposal will focus on elucidating the biochemical mechanisms by which FoxA proteins activate the apoA1 gene. It is expected that a detailed understanding at this level will provide the necessary groundwork for future development of therapeutic strategies to address metabolic diseases that result from defective expression of relevant genes. FoxA proteins have been suggested to function as "pioneer factors", which, being the first to bind their target sites, trigger cascades of gene activity. Gene activation is a multistep process that is partly dependent on covalent modifications of distinct residues in nucleosomal histones. Recently, it was found that FoxAl binds only to target sites that are enriched with nucleosomes bearing histone H3 that is mono- or dimethylated on lysines at position 4 (H3K4). Further, there are preliminary data indicating that FoxA binds strongly to coactivator complexes containing H3K4 methyltransferases. Because maximal gene activity correlates best with a trimethylated state of H3K4, it is hypothesized that FoxA mediates the acquisition of this state by recruiting the appropriate coactivators. To test this hypothesis, this proposal will utilize both biochemical and cell- based assays. Biochemical approaches will include isolation of the FoxA-recruited methyltransferase coactivator complexes, reconstitution of FoxA-dependent function from chromatinized templates in in vitro transcription reactions, and dissection of the underlying mechanistic steps. Cell-based methods (ChIP and siRNA) will be applied to a model cell culture system for hepatocytes. The main aims are therefore designed to determine the precise mechanisms whereby such coactivators are recruited and what the consequences of their recruitment are. PUBLIC HEALTH RELEVANCE: Given the widespread incidence of the metabolic diseases such as heart disease, hypertension, and diabetes, it is critical to understand how the genes whose defective expression has been tied to these diseases are normally controlled in the cell. This proposal will focus on a protein that controls expression of these genes in the liver and aim to work out the detailed mechanisms of its action.